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Studies on ACTH-induced melanocyte formation: Effect of colchicine
Experimental
Cell Kesectrch 25, 454-484 (1961)
BRIEF
STUDIES
ON ACTH-INDUCED EFFECT KI-HAN
Department
NOTES
KI.M,
MELANOCYTE
OF COLCHICINE T. T. TCHEN
FORMATION: 1
and FLJNAN
of Chemistry, Wayne State University, and Department Henry Ford Hospital, Detroit, Michigan, U.S.A.
HU of Dermatology,
Received July 14, 1961
IT
is well established that during embryonic development of the higher animals. certain cells of the neural crest migrate to the skin and become propigment cells (or melanoblasts). (The evidence has been summarized by DuShane 121.) At a later stage of development, these propigment cells transform into melanocytes. In the completely xanthic goldfish, this normal sequence of development is arrested at the propigment cell stage and no melanocytes are present in the skin. The formation of melanocytes can, however, be readily induced by either the application of stress to whole fish or by the addition of ACTH to cultures of xanthic tailfin [I, 31. We wish to report in this communication the results of some recent studies on the effect of colchicine on this ACTH-induced melanocyte formation. These results suggest that ACTH initiates nuclear division in the propigment cells and thus leads to cellular differentiation and the formation of melanocytes. Experimental and results.---Explants of xanthic goldfish tail were cut, attached to slides by means of plasma clots and incubated as previously described [3]. \\:hen ACTH was used, it was added to the medium at a concentration of 0.2 I.U./ml. The total incubation time was always 72 hr. In several experiments, it was found that colchicine at a concentration of 0.3 pg/ml or more completely inhibited the ACTHinduced formation of melanocytes. 0.1 pg/ml of colchicine gave marginal and variable response, whereas 0.01 pg/ml had no effect. In separate experiments, it was established that colchicine at 40 ,ug/ml did not inhibit the action of tyrosinase partially purified from goldfish skin. (Assay method and purification procedure will be published elsewhere.) [4, 51. The inhibitory effect of colchicine on the formation of melanocyte is thus not due to inhibition of the enzyme responsible for melanin formation. The possibility that colchicine may have caused permanent damage to the cells was Explants were incubated in the presence eliminated by the following experiment. ol’ ACTH and colchicine for certain periods of time and then transferred to and incubated in fresh medium containing ACTH hut no colchicine. The results are shown in Table I. If the explants were pre-incubated with ACTH for 12 hr or more, the addition 01 1 Supported 1)~ (;ranIs (;Y-2234 ant1 H-4130 from Ihc 9ational
Tnstilutes oi’ Health.
Studies on ACTH-induced TABLE
I. Pre-incubation
with colchicine. Explants with the following number of melanorytes
Hours of incubation with .\CTH f Colchicinea .%(:TH
None
72
1-8
9-l 6
>, 16
4 72
-
9
12
60
-
9
24
-18
7
36
36
4
IL In this and in subsequent
cspwitttents
TABLE
IIours
of incubation
II.
the concentration
Pre-incubation
of colchicinc
None
66
9
61)
1
6
24
1x
3
1
36 -
III.
2
1 5
3
1
4
11
-
Jlinimum
incubation
>16
-
for melanocyte formation.
time wifh ACTH
1:splants with the following nutnber of melanocytes
Hours incubation \Vilh A1lTII
9-l 6
1-8
6
72
1 pg/ml.
with ACTH.
12 36
was always
Explants with the following number of mclanocytes
with
I-\CTH + Colchicinc
AC’I‘H
TAULE
455
melnnocyte formation: effect of colchicine
WYthout .\CTH
Sent
72
1-8
11-16
-
36
36
2-1
4x
21
6
i lti
3 8
9
12
60
10
7
5
s
64
19
5
1
-1
68
10
72
32
:30 1
456
Ki-Han Kim, T. T. Tchen and Funan Hu TABLE IV. Pre-incubation
Hous of incubation ACTH + Colchicine Basal medium 72 60 54 48 36
12
18 24 36 72
with ACTH
o-2
24 -
and colchicine.
Explants with the following number of melanocgtes 3-x 9-l 6
5 2
16
77
7
10
9
7 3
13 20
22
72 hr with ACTH
24
colchicine can no longer inhibit the appearance of melanocytes. This is shown in Table II. It is of interest to compare this table with Table III. In this series of experiments, explants were incubated first with ACTH and then transferred to basal culture medium without ACTH. A minimum of 8-12 hr of contact with ACTH is required for the appearance of melanocytes. The minimum time required here is approximately the same as, or maybe a little less than, the time required for the cells to become insensitive to colchicine. These experiments show that in the transformation of promelanocytes (melanoblasts) to melanocytes, only the early phase requires ACTH. Cells in the late phase no longer require ACTH and are also insensitive to colchicine. We next undertook to determine whether the ACTH-requiring process is the same as the one that is inhibited by colchicine. For this purpose, explants were incubated with both ACTH and colchicine for 12, 24 and 36 hr and then transferred to basal medium with neither ACTH nor colchicine and further incubated for 60, 48 and 36 hr respectively. As shown in Table IV, melanocytes were formed in all cases, indicating that ACTH was capable of initiating the differentiation of melanoblasts in the presence of colchicine. Discussion.-From the above experiments, the following conclusions may be drawn. (1) The early phase of the melanoblast-melanocyte transformation requires ACTH. This phase lasts for a minimum of approximately 12 hr and is insensitive to colchicine. (2) The late phase of the melanoblast-melanocyte transformation is neither ACTH-dependent nor colchicine-sensitive. (3) Somewhere between these two phases, colchicine exerts an inhibitory effect. One explanation of these observations is that melanoblasts undergo nuclear division in the presence of ACTH and thus lead to the appearance of melanocytes. Colchicine inhibits this process by arresting mitosis at metaphase. Alternatively, one can postulate a process of differentiation of melanoblasts to melanocytes without nuclear division. One would then have to account for the effect of colchicine by some hitherto unknown physiological effect of this compound. Further experiments are in progress to explore these two possibilities.
Experimental
Cell liesearch 25
Studies on ACTH-induced
melanocyte formation: effect of colchicine
457
REFERENCES 1. CHAVIX, VT., J. Exptl. Zool. 133, 1 (1956). 2. DUSHANE, G. P., in The Biology of Melanoma, vol. 3, p. 1. New York Academy 1948. 3. Hu, F. and CHAVIN, W., .J. Inuest. DermatoI. 34, 377 (1960). 4. KIM, I<. and TCHEX, T. T., Uiochim. et Biophys. Beta. In press. 5. KIM, I<., TCHEN, T. T., CHAWS, W. and Hu, l:., Federation Proc. 21, 223 (1961).
THE
ACTIVATION
OF NUCLEAR
DURING J. MALEC,
L. KORNACKA,
Department
NUCLEOPROTEINS
PHAGOCYTOSIS
M. WOJNAROWSKA
of Biochemistry,
of Sciences,
Institute
of Haematology,
and K. ZAKRZEWSKI Warsaw, Poland
Received July 28, 1961
R~GOCYTOSIS PZ
brings about a general intensification of the metabolism of macrophagic cells [2, 13, 141, as well as the liberation of substances capable of killing the engulfed microorganisms [6]. It remains, however, unknown whether the phagocytic stimulus is followed by a specific anabolic event leading to the production of substances reacting with microorganisms. This problem has now been studied by assaying the incorporation of some of the precursors of RNA and of proteins into various subcellular fractions isolated from the phagocytosing human circulatory leucocytes. The leucocytes were isolated from human plasma and suspended in the nutrient medium as previously described [9]. For phagocytosis, heat-killed Sfaph. albus were added in the proportion of 5-10 bacteria per leucocyte, and the micture incubated at 37°C for l-2 hr. The cells were then washed, disrupted in a Potter-Elvehjem type homogenizer and fractionated in 0.25 M and 0.34 M sucrose. Three fractions were collected: nuclei, “mitochondria” which contained also coarse granules, and “cell sap” which contained also microsomes. The nuclei were additionally washed with 0.01 jlf and 0.05 M citric acid. After removal of small molecular substances with trichloracetic acid (TCA) and of lipids with organic solvents, the proteins were separated from nucleic acids by TCA extraction at 90°C for 15 min. Protein was determined by the method of Lowry [8] and prepared for radioactive assays according to Simkin and Work [15]. RNA was determined by the method of Mejbaum [ll]. In the 3ZP experiments the incorporation of phosphorus into RNA was determined after electrophoretic separation of mononucleotides [lo]. In the 1% erotic acid experiments, the counts found in the hot TCA extract were taken to be entirely due to the incorporation of this precursor into RNA, after it has been ascertained that erotic acid is not incorporated into DNA of mature human leucocytes. The incorporation of phosphate labelled with 32P into nuclear RNA was markedly stimulated by phagocytosis, but not the incorporation of 14C glycine into nuclear proteins, as shown in Tables I and II. Such difference might he expected in cases Experimenlal
Cell Research 25
Cell Kesectrch 25, 454-484 (1961)
BRIEF
STUDIES
ON ACTH-INDUCED EFFECT KI-HAN
Department
NOTES
KI.M,
MELANOCYTE
OF COLCHICINE T. T. TCHEN
FORMATION: 1
and FLJNAN
of Chemistry, Wayne State University, and Department Henry Ford Hospital, Detroit, Michigan, U.S.A.
HU of Dermatology,
Received July 14, 1961
IT
is well established that during embryonic development of the higher animals. certain cells of the neural crest migrate to the skin and become propigment cells (or melanoblasts). (The evidence has been summarized by DuShane 121.) At a later stage of development, these propigment cells transform into melanocytes. In the completely xanthic goldfish, this normal sequence of development is arrested at the propigment cell stage and no melanocytes are present in the skin. The formation of melanocytes can, however, be readily induced by either the application of stress to whole fish or by the addition of ACTH to cultures of xanthic tailfin [I, 31. We wish to report in this communication the results of some recent studies on the effect of colchicine on this ACTH-induced melanocyte formation. These results suggest that ACTH initiates nuclear division in the propigment cells and thus leads to cellular differentiation and the formation of melanocytes. Experimental and results.---Explants of xanthic goldfish tail were cut, attached to slides by means of plasma clots and incubated as previously described [3]. \\:hen ACTH was used, it was added to the medium at a concentration of 0.2 I.U./ml. The total incubation time was always 72 hr. In several experiments, it was found that colchicine at a concentration of 0.3 pg/ml or more completely inhibited the ACTHinduced formation of melanocytes. 0.1 pg/ml of colchicine gave marginal and variable response, whereas 0.01 pg/ml had no effect. In separate experiments, it was established that colchicine at 40 ,ug/ml did not inhibit the action of tyrosinase partially purified from goldfish skin. (Assay method and purification procedure will be published elsewhere.) [4, 51. The inhibitory effect of colchicine on the formation of melanocyte is thus not due to inhibition of the enzyme responsible for melanin formation. The possibility that colchicine may have caused permanent damage to the cells was Explants were incubated in the presence eliminated by the following experiment. ol’ ACTH and colchicine for certain periods of time and then transferred to and incubated in fresh medium containing ACTH hut no colchicine. The results are shown in Table I. If the explants were pre-incubated with ACTH for 12 hr or more, the addition 01 1 Supported 1)~ (;ranIs (;Y-2234 ant1 H-4130 from Ihc 9ational
Tnstilutes oi’ Health.
Studies on ACTH-induced TABLE
I. Pre-incubation
with colchicine. Explants with the following number of melanorytes
Hours of incubation with .\CTH f Colchicinea .%(:TH
None
72
1-8
9-l 6
>, 16
4 72
-
9
12
60
-
9
24
-18
7
36
36
4
IL In this and in subsequent
cspwitttents
TABLE
IIours
of incubation
II.
the concentration
Pre-incubation
of colchicinc
None
66
9
61)
1
6
24
1x
3
1
36 -
III.
2
1 5
3
1
4
11
-
Jlinimum
incubation
>16
-
for melanocyte formation.
time wifh ACTH
1:splants with the following nutnber of melanocytes
Hours incubation \Vilh A1lTII
9-l 6
1-8
6
72
1 pg/ml.
with ACTH.
12 36
was always
Explants with the following number of mclanocytes
with
I-\CTH + Colchicinc
AC’I‘H
TAULE
455
melnnocyte formation: effect of colchicine
WYthout .\CTH
Sent
72
1-8
11-16
-
36
36
2-1
4x
21
6
i lti
3 8
9
12
60
10
7
5
s
64
19
5
1
-1
68
10
72
32
:30 1
456
Ki-Han Kim, T. T. Tchen and Funan Hu TABLE IV. Pre-incubation
Hous of incubation ACTH + Colchicine Basal medium 72 60 54 48 36
12
18 24 36 72
with ACTH
o-2
24 -
and colchicine.
Explants with the following number of melanocgtes 3-x 9-l 6
5 2
16
77
7
10
9
7 3
13 20
22
72 hr with ACTH
24
colchicine can no longer inhibit the appearance of melanocytes. This is shown in Table II. It is of interest to compare this table with Table III. In this series of experiments, explants were incubated first with ACTH and then transferred to basal culture medium without ACTH. A minimum of 8-12 hr of contact with ACTH is required for the appearance of melanocytes. The minimum time required here is approximately the same as, or maybe a little less than, the time required for the cells to become insensitive to colchicine. These experiments show that in the transformation of promelanocytes (melanoblasts) to melanocytes, only the early phase requires ACTH. Cells in the late phase no longer require ACTH and are also insensitive to colchicine. We next undertook to determine whether the ACTH-requiring process is the same as the one that is inhibited by colchicine. For this purpose, explants were incubated with both ACTH and colchicine for 12, 24 and 36 hr and then transferred to basal medium with neither ACTH nor colchicine and further incubated for 60, 48 and 36 hr respectively. As shown in Table IV, melanocytes were formed in all cases, indicating that ACTH was capable of initiating the differentiation of melanoblasts in the presence of colchicine. Discussion.-From the above experiments, the following conclusions may be drawn. (1) The early phase of the melanoblast-melanocyte transformation requires ACTH. This phase lasts for a minimum of approximately 12 hr and is insensitive to colchicine. (2) The late phase of the melanoblast-melanocyte transformation is neither ACTH-dependent nor colchicine-sensitive. (3) Somewhere between these two phases, colchicine exerts an inhibitory effect. One explanation of these observations is that melanoblasts undergo nuclear division in the presence of ACTH and thus lead to the appearance of melanocytes. Colchicine inhibits this process by arresting mitosis at metaphase. Alternatively, one can postulate a process of differentiation of melanoblasts to melanocytes without nuclear division. One would then have to account for the effect of colchicine by some hitherto unknown physiological effect of this compound. Further experiments are in progress to explore these two possibilities.
Experimental
Cell liesearch 25
Studies on ACTH-induced
melanocyte formation: effect of colchicine
457
REFERENCES 1. CHAVIX, VT., J. Exptl. Zool. 133, 1 (1956). 2. DUSHANE, G. P., in The Biology of Melanoma, vol. 3, p. 1. New York Academy 1948. 3. Hu, F. and CHAVIN, W., .J. Inuest. DermatoI. 34, 377 (1960). 4. KIM, I<. and TCHEX, T. T., Uiochim. et Biophys. Beta. In press. 5. KIM, I<., TCHEN, T. T., CHAWS, W. and Hu, l:., Federation Proc. 21, 223 (1961).
THE
ACTIVATION
OF NUCLEAR
DURING J. MALEC,
L. KORNACKA,
Department
NUCLEOPROTEINS
PHAGOCYTOSIS
M. WOJNAROWSKA
of Biochemistry,
of Sciences,
Institute
of Haematology,
and K. ZAKRZEWSKI Warsaw, Poland
Received July 28, 1961
R~GOCYTOSIS PZ
brings about a general intensification of the metabolism of macrophagic cells [2, 13, 141, as well as the liberation of substances capable of killing the engulfed microorganisms [6]. It remains, however, unknown whether the phagocytic stimulus is followed by a specific anabolic event leading to the production of substances reacting with microorganisms. This problem has now been studied by assaying the incorporation of some of the precursors of RNA and of proteins into various subcellular fractions isolated from the phagocytosing human circulatory leucocytes. The leucocytes were isolated from human plasma and suspended in the nutrient medium as previously described [9]. For phagocytosis, heat-killed Sfaph. albus were added in the proportion of 5-10 bacteria per leucocyte, and the micture incubated at 37°C for l-2 hr. The cells were then washed, disrupted in a Potter-Elvehjem type homogenizer and fractionated in 0.25 M and 0.34 M sucrose. Three fractions were collected: nuclei, “mitochondria” which contained also coarse granules, and “cell sap” which contained also microsomes. The nuclei were additionally washed with 0.01 jlf and 0.05 M citric acid. After removal of small molecular substances with trichloracetic acid (TCA) and of lipids with organic solvents, the proteins were separated from nucleic acids by TCA extraction at 90°C for 15 min. Protein was determined by the method of Lowry [8] and prepared for radioactive assays according to Simkin and Work [15]. RNA was determined by the method of Mejbaum [ll]. In the 3ZP experiments the incorporation of phosphorus into RNA was determined after electrophoretic separation of mononucleotides [lo]. In the 1% erotic acid experiments, the counts found in the hot TCA extract were taken to be entirely due to the incorporation of this precursor into RNA, after it has been ascertained that erotic acid is not incorporated into DNA of mature human leucocytes. The incorporation of phosphate labelled with 32P into nuclear RNA was markedly stimulated by phagocytosis, but not the incorporation of 14C glycine into nuclear proteins, as shown in Tables I and II. Such difference might he expected in cases Experimenlal
Cell Research 25